Gangliosides are a class of glycosphingolipids, having one or more sialic acid residues, and are found abundantly in the cerebral and nervous tissue of humans and animals. The monosialoganglioside GM1 is known for a number of pharmaceutical applications, particularly in the repair and treatment of disorders of the central and peripheral nervous systems.
Gangliosides are conveniently extracted from bovine or porcine cerebral tissue according to U.S. Pat. No. 4,849,413. In order to be suitable for pharmaceutical applications, the monosialoganglioside GM1 must then be isolated and purified.
It is known to treat the extracted lipid mixture by chemical or enzymatic methods to transform other ganglioside components to the monosialoganglioside GM1, in order to increase the yield of monosialoganglioside GM1. Such methods include acid hydrolysis with a weak acid, such as described in CN 1353112, or enzymatic hydrolysis using a sialidase, for instance as described in U.S. Pat. No. 5,296,360.
Further purification of such GM1 enhanced lipidic mixture by exclusion chromatography using a chloroform:methanol:water 60:30:4.5 eluent is described in EP 0 150 712. EP 0 489 352 describes purification of a GM1 enhanced lipidic mixture by ultra-filtration of a solution of the lipidic mixture with alpha-cyclo-dextrin, followed by extraction of GM1 by solvent extraction with ethanol. It is reported that GM1 may be obtained with a purity of 95%.
Such processes have previously been shown to have drawbacks with respect to the purity and yield of GM1, and with respect to cost, efficiency and effectiveness when applied on an industrial scale.
For pharmaceutical applications it is required to produce the ganglioside GM1 at high purity. Accordingly, there remains an ongoing need for processes for obtaining the ganglioside GM1 at high-purity.
The inventors of present application have surprisingly found that the ganglioside GM1 may be effectively separated from other gangliosides by a process based on ion-exchange chromatography.
According to the present invention, it has been found that the ganglioside GM1 may be prepared at high purity by a process wherein GM1 is separated from a lipidic mixture containing the monosialoganglioside GM1 as the main ganglioside component by ion exchange column-chromatography using an eluent comprising potassium or caesium ions.
According to the present invention there is provided a process for the isolation and purification of the ganglioside GM1 according to claim 1.
According to a preferred embodiment of the present invention there is provided a process comprising the general steps of:
(a) separating GM1 from a lipidic mixture containing the monosialoganglioside GM1 as the main ganglioside component by ion exchange column-chromatography using an eluent comprising potassium or caesium ions,
(b) recovery of the solute from the eluted solution,
(c) diafiltration of an aqueous solution of the recovered solute,
(d) addition of a sodium salt and diafiltration of the resultant solution, and
(e) recovery of GM1.
Advantageously the process of the present invention allows for the preparation of the monoganglioside GM1 at high purity. According to the present invention the monosialoganglioside GM1 may be prepared with a purity of higher than 98%, even higher than 99.0% and even 99.9%.
Further, the process of the present invention advantageously uses simple steps, is cost-efficient and is suitable for application on an industrial scale.
Other objects and advantages of the present invention will be apparent from the claims and from the following detailed description and examples.